A number of therapeutic agents comprising targeting ligands linked to a drug contain one or more linkers (e.g. disulfide-based linkers) placed between the targeting ligand and the therapeutically active drug. The targeting ligand allows for specific binding to cells that express a receptor for the ligand (e.g. cancer cells), resulting in delivery of the therapeutically active drug to the cells of interest. The disulfide linker allows for release of the drug inside the cell when the targeting ligand-drug conjugate is internalized and is exposed to the reducing environment inside the cells. For example, specific binding of folate-drug conjugates to the folate receptor (FR) on cancer cells or inflammatory cells allows for targeted delivery and specific therapeutic activity directed to the cancer cells or to sites of inflammation.
Folate is a member of the B family of vitamins and plays an essential role in cell survival by participating in the biosynthesis of nucleic acids and amino acids. This essential vitamin is also a high affinity ligand that enhances the specificity of conjugated anti-cancer drugs by targeting, for example, FR-positive cancer cells or inflammatory cells. The FR, a glycosylphosphatidyl-inositol anchored protein, can actively internalize bound folates and folate conjugated compounds via receptor-mediated endocytosis. It has been found that the FR is up-regulated in more than 90% of non-mucinous ovarian carcinomas, and is also found on inflammatory cells. The FR is also found at high to moderate levels in kidney, brain, lung, and breast carcinomas while it occurs at low levels in most normal tissues. FR density appears to increase as the stage of the cancer becomes more advanced. Folate-targeted drug conjugates have been developed and are being tested in clinical trials as cancer therapeutics, and in pre-clinical testing as therapeutics for inflammatory diseases.
Generally, the disulfide linker of targeted conjugates remains relatively stable in circulation, but rapidly breaks down once the conjugate enters the reductive endocytic process within a targeted cell. However, the possibility exists that a disulfide linker may be broken down outside cells, resulting in release of the drug and non-specific toxicity. The current inventors have hypothesized that instability of the disulfide linker in targeted conjugates may contribute to non-specific toxicity, and that the presence of extracellular thiols may play a role in the instability of the disulfide linker with resulting non-specific toxicity.
Accordingly, two different approaches were undertaken to evaluate the impact of extracellular thiols on non-specific activity of targeted conjugates with disulfide linkers. First, ligand conjugates and thiol inhibitors were tested together to evaluate the effects of co-treatment of cells with ligand conjugates and thiol inhibitors on the nonspecific uptake of the drug into the target cells and on non-ligand specific activity. Second, ligand conjugates and system xc− inhibitors were tested together to evaluate the effects of co-treatment of cells with ligand conjugates and system xc− inhibitors on the nonspecific uptake of the drug into the target cells and on non-ligand specific activity.
Surprisingly, the inventors have found that extracellular thiols (e.g. cysteine) contribute to non-specific toxicity of targeted conjugates containing one or more disulfide linkers, especially when the extracellular ligand conjugate concentration is exceedingly high. Thus, co-administration of thiol inhibitors or system xc− inhibitors to patients along with targeted conjugates with disulfide linkers may decrease non-specific toxicity and increase efficacy of targeted therapeutics.
In one embodiment, a method of treatment of a disease is provided. The method comprises administering a ligand conjugate to a patient, wherein the ligand conjugate comprises a disulfide linkage; and administering a thiol inhibitor to the patient.
In another embodiment, use of a ligand conjugate in combination with a thiol inhibitor for the treatment of a disease is described, wherein the disease is cancer or inflammation, and wherein the ligand conjugate comprises a disulfide linkage.
In another embodiment, use of a ligand conjugate for the manufacture of a medicament for the treatment of a disease is described, wherein the disease is cancer or inflammation, and wherein the treatment comprises treating a patient with the ligand conjugate in combination with a thiol inhibitor, wherein the ligand conjugate comprises a disulfide linkage.
In another embodiment, a kit is provided. The kit comprises a ligand conjugate and one or more thiol inhibitors, wherein the ligand conjugate comprises a disulfide linkage.
In another embodiment, an in vitro assay for identifying a ligand conjugate suitable for co-administration with a thiol inhibitor to a patient is provided. The assay comprises the steps of a) adding the ligand conjugate to the culture medium of a first sample of cultured cells, wherein the ligand conjugate comprises a disulfide linkage; b) adding the thiol inhibitor to the culture medium of the first sample of cultured cells to provide a test sample; c) adding the ligand conjugate to the culture medium of a second sample of cultured cells to provide a control sample; d) measuring the non-ligand-specific activity of the ligand conjugate or the nonspecific uptake of the drug in the test sample; e) measuring the non-ligand-specific activity of the ligand conjugate or the nonspecific uptake of the drug in the control sample; and f) determining that the ligand conjugate is suitable for co-administration to the patient with the thiol inhibitor if the non-ligand-specific activity of the ligand conjugate and/or the nonspecific uptake of the drug are decreased in the test sample relative to the control sample.
In yet another embodiment, a method of treatment of a disease is provided. The method comprises administering a ligand conjugate to a patient, wherein the ligand conjugate comprises a disulfide linkage; and administering a system xc− inhibitor to the patient.
In another embodiment, use of a ligand conjugate in combination with a system xc− inhibitor for the treatment of a disease is described, wherein the disease is cancer or inflammation, and wherein the ligand conjugate comprises a disulfide linkage.
In another embodiment, use of a ligand conjugate for the manufacture of a medicament for the treatment of a disease wherein the disease is cancer or inflammation is described, and wherein the treatment comprises treating a patient with the ligand conjugate in combination with a system xc− inhibitor, wherein the ligand conjugate comprises a disulfide linkage.
In another embodiment, a kit is provided. The kit comprises a ligand conjugate and one or more system xc− inhibitors, wherein the ligand conjugate comprises a disulfide linkage.
In another embodiment, an in vitro assay for identifying a ligand conjugate suitable for co-administration to a patient with a system xc− inhibitor is provided. The assay comprises the steps of a) adding the ligand conjugate to the culture medium of a first sample of cultured cells, wherein the ligand conjugate comprises a disulfide linkage; b) adding the system xc− inhibitor to the culture medium of the first sample of cultured cells to provide a test sample; c) adding the ligand conjugate to the culture medium of a second sample of cultured cells to provide a control sample; d) measuring the non-ligand-specific activity of the ligand conjugate or the nonspecific uptake of the drug in the test sample; e) measuring the non-ligand-specific activity of the ligand conjugate or the nonspecific uptake of the drug in the control sample; and f) determining that the ligand conjugate is suitable for co-administration to the patient with the system xc− inhibitor if the non-ligand-specific activity of the ligand conjugate and/or the nonspecific uptake of the drug are decreased in the test sample relative to the control sample. Any of the embodiments described in the following clause list are considered to be part of the invention.                1. A method of treatment of a disease, the method comprising the steps of:                    administering a ligand conjugate to a patient, wherein the ligand conjugate comprises a disulfide linkage; and            administering a thiol inhibitor to the patient.                        2. The method of clause 1, wherein the disease is cancer or inflammation.        3. Use of a ligand conjugate in combination with a thiol inhibitor for the treatment of a disease wherein the disease is cancer or inflammation, and wherein the ligand conjugate comprises a disulfide linkage.        4. Use of a ligand conjugate for the manufacture of a medicament for the treatment of a disease wherein the disease is cancer or inflammation, and wherein the treatment comprises treating a patient with the ligand conjugate in combination with a thiol inhibitor, wherein the ligand conjugate comprises a disulfide linkage.        5. The method or use of any one of clauses 1 to 4, wherein the disease is inflammation.        6. The method or use of any one of clauses 1 to 4, wherein the disease is cancer.        7. The method or use of clause 6, wherein the cancer comprises a primary tumor.        8. The method or use of clause 6, wherein the cancer comprises metastatic tumor cells.        9. The method or use of any one of clauses 1 to 8, wherein the ligand is folate.        10. The method or use of any one of clauses 1 to 8, wherein the ligand is an antibody.        11. The method or use of any one of clauses 1 to 8, wherein the ligand conjugate is of the formula BLDx, wherein B is a cell surface receptor targeting ligand, D is an independently selected drug, x is an integer selected from 1, 2, 3, 4 and 5; and L is a releasable polyvalent linker comprising a thiol reactive linkage; or a pharmaceutically acceptable salt thereof.        12. The method or use of clause 11, wherein B is folate.        13. The method or use of clause 11, wherein B is D-folate.        14. The method or use of clause 11, wherein B is L-folate.        15. The method or use of clause 11, wherein B is a PSMA binding ligand.        16. The method or use of clause 11, wherein B is a radical of the formula        
                17. The method or use of clause 11, wherein B is a radical of the formula        
                18. The method or use of clause 11, wherein B is a radical of the formula        
                19. The method or use of any one of clauses 11 to 18, wherein the thiol reactive linkage is a disulfide linkage.        20. The method or use of any one of clauses 11 to 19, wherein L comprises a cysteine disulfide diradical.        21. The method or use of any one of clauses 11 to 20, wherein L further comprises one or more divalent hydrophilic radicals.        22. The method or use of any one of clauses 11 to 21, wherein D is a cytotoxic agent.        23. The method or use of any one of clauses 11 to 22, wherein D is a cancer treating agent.        24. The method or use of any one of clauses 11 to 22, wherein D is an anti-inflammatory agent.        25. The method or use of any one of clauses 11 to 24, wherein D is a vinca alkaloid.        26. The method or use of any one of clauses 11 to 24, wherein D is desacetylvinblastine monohydrazide.        27. The method or use of any one of clauses 11 to 24, wherein D is a tubulysin.        28. The method or use of any one of clauses 11 to 24, wherein D is tubulysin A.        29. The method or use of any one of clauses 11 to 24, wherein D is tubulysin B.        30. The method or use of any one of clauses 11 to 24, wherein D is tubulysin A hydrazide.        31. The method or use of any one of clauses 11 to 24, wherein D is tubulysin B hydrazide.        32. The method or use of any one of clauses 11 to 24, wherein D is an antifolate.        33. The method or use of any one of clauses 11 to 24, wherein D is an aminopterin.        34. The method or use of any one of clauses 11 to 24, wherein D is a rapamycin.        35. The method or use of any one of clauses 11 to 24, wherein D is a mitomycin.        36. The method or use of any one of clauses 11 to 24, wherein D is a taxane.        37. The method or use of any one of clauses 11 to 24, wherein D is a doxorubicin.        38. The method or use of any one of clauses 1 to 9 or 11 to 37, wherein the ligand conjugate is a folate conjugate.        39. The method or use of clause 38, wherein the folate conjugate is        
                40. The method or use of clause 38, wherein the folate conjugate is        
                41. The method or use of any one of clauses 1 to 40, wherein the thiol inhibitor is selected from the group consisting of 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB); maleimides (e.g. N-maleoyl-β-alanine (N-(2-carboxyethyl) maleimide (NCEM)); p-chloromercuribenzene sulfonate (pCMBS); 4-(N—(S-glutathionylacetyl)amino) phenylarsonous acid (GSAO); 2,2′-dithio-bis-ethanesulfonate (dimesna); oxidized glutathione (GSSG); vinyl sulfone compounds (e.g. methoxy-PEG5000-vinylsulfone); epigallocatechin gallate (EGCG); and 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS).        42. The method or use of any one of clauses 1 to 41, wherein the thiol inhibitor is DTNB.        43. The method or use of any one of clauses 1 to 41, wherein the thiol inhibitor is a maleimide.        44. The method or use of any one of clauses 1 to 41, wherein the thiol inhibitor is NCEM.        45. The method or use of any one of clauses 1 to 41, wherein the thiol inhibitor is pCMBS.        46. The method or use of any one of clauses 1 to 41, wherein the thiol inhibitor is GSAO.        47. The method or use of any one of clauses 1 to 41, wherein the thiol inhibitor is dimesna.        48. The method or use of any one of clauses 1 to 41, wherein the thiol inhibitor is GSSG.        49. The method or use of any one of clauses 1 to 41, wherein the thiol inhibitor is a vinyl sulfone compound.        50. The method or use of any one of clauses 1 to 41, wherein the thiol inhibitor is methoxy-PEG5000-vinylsulfone.        51. The method or use of any one of clauses 1 to 41, wherein the thiol inhibitor is EGCG.        52. The method or use of any one of clauses 1 to 41, wherein the thiol inhibitor is AMS.        53. The method or use of any one of clauses 1 to 52, wherein the ligand conjugate and the thiol inhibitor are in parenteral dosage forms.        54. The method or use of clause 53, wherein the dosage forms are independently selected from the group consisting of intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, and intrathecal.        55. The method or use of any one of clauses 1 to 54, wherein the ligand conjugate is in a composition and the thiol inhibitor is in a composition and wherein the compositions further comprise pharmaceutically acceptable carriers.        56. The method or use of clause 55, wherein the pharmaceutically acceptable carriers are liquid carriers.        57. The method or use of clause 56, wherein the liquid carriers are independently selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof.        58. The method or use of any one of clauses 1 to 57, wherein the ligand conjugate and the thiol inhibitor are administered in therapeutically effective amounts.        59. The method or use of clause 58, wherein the effective amounts range from about 1 μg/m2 to about 500 mg/m2 of body surface area.        60. The method or use of clause 58, wherein the effective amounts range from about 1 μg/m2 to about 300 mg/m2 of body surface area.        61. The method or use of clause 58, wherein the effective amounts range from about 10 μg/kg to about 100 μg/kg of patient body weight.        62. The method or use of any one of clauses 1 to 61, wherein the ligand conjugate and the thiol inhibitor are in sterile containers or packages.        63. The method or use of any one of clauses 1 to 62, wherein the ligand conjugate and the thiol inhibitor have a purity of at least 90% based on weight percentage.        64. The method or use of any one of clauses 4 to 63, wherein the ligand conjugate is in the form of a reconstitutable lyophilizate.        65. The method or use of any one of clauses 1 to 64, wherein the ligand conjugate and the thiol inhibitor are in sterile, pyrogen-free aqueous solutions.        66. A kit comprising a ligand conjugate and one or more thiol inhibitors, wherein the ligand conjugate comprises a disulfide linkage.        67. The kit of clause 66, wherein the ligand is folate.        68. The kit of clause 66, wherein the ligand is an antibody.        69. The kit of clause 66, wherein the ligand conjugate is of the formula BLDx, wherein B is a cell surface receptor targeting ligand, D is an independently selected drug, x is an integer selected from 1, 2, 3, 4 and 5; and L is a releasable polyvalent linker comprising a thiol reactive linkage; or a pharmaceutically acceptable salt thereof.        70. The kit of clause 69, wherein B is folate.        71. The kit of clause 69, wherein B is D-folate.        72. The kit of clause 69, wherein B is L-folate.        73. The kit of clause 69, wherein B is a PSMA binding ligand.        74. The kit of clause 69, wherein B is a radical of the formula        
                75. The kit of clause 69, wherein B is a radical of the formula        
                76. The kit of clause 69, wherein B is a radical of the formula        
                77. The kit of any one of clauses 69 to 76, wherein the thiol reactive linkage is a disulfide linkage.        78. The kit of any one of clauses 69 to 77, wherein L comprises a cysteine disulfide diradical.        79. The kit of any one of clauses 69 to 78, wherein L further comprises one or more divalent hydrophilic radicals.        80. The kit of any one of clauses 69 to 79, wherein D is a cytotoxic agent.        81. The kit of any one of clauses 69 to 80, wherein D is a cancer treating agent.        82. The kit of any one of clauses 69 to 80, wherein D is an anti-inflammatory agent.        83. The kit of any one of clauses 69 to 82, wherein D is a vinca alkaloid.        84. The kit of any one of clauses 69 to 82, wherein D is desacetylvinblastine monohydrazide.        85. The kit of any one of clauses 69 to 82, wherein D is a tubulysin.        86. The kit of any one of clauses 69 to 82, wherein D is tubulysin A.        87. The kit of any one of clauses 69 to 82, wherein D is tubulysin B.        88. The kit of any one of clauses 69 to 82, wherein D is tubulysin A hydrazide.        89. The kit of any one of clauses 69 to 82, wherein D is tubulysin B hydrazide.        90. The kit of any one of clauses 69 to 82, wherein D is an antifolate.        91. The kit of any one of clauses 69 to 82, wherein D is an aminopterin.        92. The kit of any one of clauses 69 to 82, wherein D is a rapamycin.        93. The kit of any one of clauses 69 to 82, wherein D is a mitomycin.        94. The kit of any one of clauses 69 to 82, wherein D is a taxane.        95. The kit of any one of clauses 69 to 82, wherein D is a doxorubicin.        96. The kit of any one of clauses 66 to 67 or 69 to 95, wherein the ligand conjugate is a folate conjugate.        97. The kit of clause 96, wherein the folate conjugate is        
                98. The kit of clause 96, wherein the folate conjugate is        
                99. The kit of any one of clauses 66 to 98, wherein the thiol inhibitor is selected from the group consisting of 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB); maleimides (e.g. N-maleoyl-β-alanine (N-(2-carboxyethyl) maleimide (NCEM)); p-chloromercuribenzene sulfonate (pCMBS); 4-(N—(S-glutathionylacetyl)amino) phenylarsonous acid (GSAO); 2,2′-dithio-bis-ethanesulfonate (dimesna); oxidized glutathione (GSSG); vinyl sulfone compounds (e.g. methoxy-PEG5000-vinylsulfone); epigallocatechin gallate (EGCG); and 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS).        100. The kit of any one of clauses 66 to 99, wherein the thiol inhibitor is DTNB.        101. The kit of any one of clauses 66 to 99, wherein the thiol inhibitor is a maleimide.        102. The kit of any one of clauses 66 to 99, wherein the thiol inhibitor is NCEM.        103. The kit of any one of clauses 66 to 99, wherein the thiol inhibitor is pCMBS.        104. The kit of any one of clauses 66 to 99, wherein the thiol inhibitor is GSAO.        105. The kit of any one of clauses 66 to 99, wherein the thiol inhibitor is dimesna.        106. The kit of any one of clauses 66 to 99, wherein the thiol inhibitor is GSSG.        107. The kit of any one of clauses 66 to 99, wherein the thiol inhibitor is a vinyl sulfone compound.        108. The kit of any one of clauses 66 to 99, wherein the thiol inhibitor is methoxy-PEG5000-vinylsulfone.        109. The kit of any one of clauses 66 to 99, wherein the thiol inhibitor is EGCG.        110. The kit of any one of clauses 66 to 99, wherein the thiol inhibitor is AMS.        111. The kit of any one of clauses 66 to 110, wherein the ligand conjugate and the thiol inhibitor are in parenteral dosage forms.        112. The kit of clause 111, wherein the dosage forms are independently selected from the group consisting of intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, and intrathecal.        113. The kit of any one of clauses 66 to 112, wherein the ligand conjugate is in a composition and the thiol inhibitor is in a composition and wherein the compositions further comprise pharmaceutically acceptable carriers.        114. The kit of clause 113, wherein the pharmaceutically acceptable carriers are liquid carriers.        115. The kit of clause 114, wherein the liquid carriers are independently selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof.        116. The kit of any one of clauses 66 to 115, wherein the ligand conjugate and the thiol inhibitor are in therapeutically effective amounts.        117. The kit of any one of clauses 66 to 116, wherein the ligand conjugate and the thiol inhibitor are in sterile containers or packages.        118. The kit of any one of clauses 66 to 117, wherein the ligand conjugate and the thiol inhibitor have a purity of at least 90% based on weight percentage.        119. The kit of any one of clauses 66 to 117, wherein the ligand conjugate and the thiol inhibitor have a purity of at least 95% based on weight percentage.        120. The kit of any one of clauses 66 to 119, wherein the ligand conjugate is in the form of a lyophilizate.        121. The kit of any one of clauses 66 to 120, wherein the ligand conjugate is in the form of a reconstitutable lyophilizate.        122. The kit of any one of clauses 66 to 121, wherein the ligand conjugate and the thiol inhibitor are in sterile, pyrogen-free aqueous solutions.        123. An in vitro assay for identifying a ligand conjugate suitable for co-administration to a patient with a thiol inhibitor, the assay comprising:        a) adding the ligand conjugate to the culture medium of a first sample of cultured cells, wherein the ligand conjugate comprises a disulfide linkage;        b) adding the thiol inhibitor to the culture medium of the first sample of cultured cells to provide a test sample;        c) adding the ligand conjugate to the culture medium of a second sample of cultured cells to provide a control sample;        d) measuring the non-ligand-specific activity of the ligand conjugate or the nonspecific uptake of the drug in the test sample;        e) measuring the non-ligand-specific activity of the ligand conjugate or the nonspecific uptake of the drug in the control sample; and        f) determining that the ligand conjugate is suitable for co-administration to the patient with the thiol inhibitor if the non-ligand-specific activity of the ligand conjugate and/or the nonspecific uptake of the drug are decreased in the test sample relative to the control sample.        124. The in vitro assay of clause 123 further comprising step g) administering the ligand conjugate and the thiol inhibitor to the patient.        125. The in vitro assay of clause 123 or clause 124 wherein the ligand is folate.        126. The in vitro assay of clause 123 or clause 124 wherein the ligand is an antibody.        127. The in vitro assay of clause 123 or clause 124, wherein the ligand conjugate is of the formula BLDx, wherein B is a cell surface receptor targeting ligand, D is an independently selected drug, x is an integer selected from 1, 2, 3, 4 and 5; and L is a releasable polyvalent linker comprising a thiol reactive linkage; or a pharmaceutically acceptable salt thereof.        128. The in vitro assay of clause 127, wherein B is folate.        129. The in vitro assay of clause 127, wherein B is D-folate.        130. The in vitro assay of clause 127, wherein B is L-folate.        131. The in vitro assay of clause 127, wherein B is a PSMA binding ligand.        132. The in vitro assay of clause 127, wherein B is a radical of the formula        
                133. The in vitro assay of clause 127, wherein B is a radical of the formula        
                134. The in vitro assay of clause 127, wherein B is a radical of the formula        
                135. The in vitro assay of any one of clauses 127 to 134, wherein the thiol reactive linkage is a disulfide linkage.        136. The in vitro assay of any one of clauses 127 to 135, wherein L comprises a cysteine disulfide diradical.        137. The in vitro assay of any one of clauses 127 to 136, wherein L further comprises one or more divalent hydrophilic radicals.        138. The in vitro assay of any one of clauses 127 to 137, wherein D is a cytotoxic agent.        139. The in vitro assay of any one of clauses 127 to 138, wherein D is a cancer treating agent.        140. The in vitro assay of any one of clauses 127 to 138, wherein D is an anti-inflammatory agent.        141. The in vitro assay of any one of clauses 127 to 140, wherein D is a vinca alkaloid.        142. The in vitro assay of any one of clauses 127 to 140, wherein D is desacetylvinblastine monohydrazide.        143. The in vitro assay of any one of clauses 127 to 140, wherein D is a tubulysin.        144. The in vitro assay of any one of clauses 127 to 140, wherein D is tubulysin A.        145. The in vitro assay of any one of clauses 127 to 140, wherein D is tubulysin B.        146. The in vitro assay of any one of clauses 127 to 140, wherein D is tubulysin A hydrazide.        147. The in vitro assay of any one of clauses 127 to 140, wherein D is tubulysin B hydrazide.        148. The in vitro assay of any one of clauses 127 to 140, wherein D is an antifolate.        149. The in vitro assay of any one of clauses 127 to 140, wherein D is an aminopterin.        150. The in vitro assay of any one of clauses 127 to 140, wherein D is a rapamycin.        151. The in vitro assay of any one of clauses 127 to 140, wherein D is a mitomycin.        152. The in vitro assay of any one of clauses 127 to 140, wherein D is a taxane.        153. The in vitro assay of any one of clauses 127 to 140, wherein D is a doxorubicin.        154. The in vitro assay of any one of clauses 123 to 140, wherein the ligand conjugate is a folate conjugate.        155. The in vitro assay of clause 154, wherein the folate conjugate is        
                156. The in vitro assay of clause 154, wherein the folate conjugate is        
                157. The in vitro assay of any one of clauses 123 to 156, wherein the thiol inhibitor is selected from the group consisting of 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB); maleimides (e.g. N-maleoyl-β-alanine (N-(2-carboxyethyl) maleimide (NCEM)); p-chloromercuribenzene sulfonate (pCMBS); 4-(N—(S-glutathionylacetyl)amino) phenylarsonous acid (GSAO); 2,2′-dithio-bis-ethanesulfonate (dimesna); oxidized glutathione (GSSG); vinyl sulfone compounds (e.g. methoxy-PEG5000-vinylsulfone); epigallocatechin gallate (EGCG); and 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS).        158. The in vitro assay of any one of clauses 123 to 157, wherein the thiol inhibitor is DTNB.        159. The in vitro assay of any one of clauses 123 to 157, wherein the thiol inhibitor is a maleimide.        160. The in vitro assay of any one of clauses 123 to 157, wherein the thiol inhibitor is NCEM.        161. The in vitro assay of any one of clauses 123 to 157, wherein the thiol inhibitor is pCMBS.        162. The in vitro assay of any one of clauses 123 to 157, wherein the thiol inhibitor is GSAO.        163. The in vitro assay of any one of clauses 123 to 157, wherein the thiol inhibitor is dimesna.        164. The in vitro assay of any one of clauses 123 to 157, wherein the thiol inhibitor is GSSG.        165. The in vitro assay of any one of clauses 123 to 157, wherein the thiol inhibitor is a vinyl sulfone compound.        166. The in vitro assay of any one of clauses 123 to 157, wherein the thiol inhibitor is methoxy-PEG5000-vinylsulfone.        167. The in vitro assay of any one of clauses 123 to 157, wherein the thiol inhibitor is EGCG.        168. The in vitro assay of any one of clauses 123 to 157, wherein the thiol inhibitor is AMS.        169. The in vitro assay of any one of clauses 123 to 168, wherein the cultured cells are KB cells.        170. The in vitro assay of any one of clauses 123 to 168, wherein the cultured cells are A549 cells.        171. The in vitro assay of any one of clauses 123 to 170, wherein the non-ligand-specific activity of the ligand conjugate is decreased by the thiol inhibitor.        172. The in vitro assay of clause 171, wherein the non-ligand-specific activity is cytotoxicity.        173. The in vitro assay of any one of clauses 123 to 170, wherein the nonspecific uptake of the drug is decreased by the thiol inhibitor.        174. The in vitro assay of clause 173, wherein the nonspecific uptake of the drug is measured using competition assays in the presence and absence of an excess of non-radiolabeled ligand.        175. A method of treatment of a disease, the method comprising the steps of:                    administering a ligand conjugate to a patient, wherein the ligand conjugate comprises a disulfide linkage; and            administering a system xc− inhibitor to the patient.                        176. The method of clause 175, wherein the disease is cancer or inflammation.        177. Use of a ligand conjugate in combination with a system xc− inhibitor for the treatment of a disease wherein the disease is cancer or inflammation, and wherein the ligand conjugate comprises a disulfide linkage.        178. Use of a ligand conjugate for the manufacture of a medicament for the treatment of a disease wherein the disease is cancer or inflammation, and wherein the treatment comprises treating a patient with the ligand conjugate in combination with a system xc− inhibitor, wherein the ligand conjugate comprises a disulfide linkage.        179. The method or use of any one of clauses 175 to 178, wherein the disease is inflammation.        180. The method or use of any one of clauses 175 to 178, wherein the disease is cancer.        181. The method or use of clause 180, wherein the cancer comprises a primary tumor.        182. The method or use of clause 180, wherein the cancer comprises metastatic tumor cells.        183. The method or use of any one of clauses 175 to 182, wherein the ligand is folate.        184. The method or use of any one of clauses 175 to 182, wherein the ligand is an antibody.        185. The method or use of any one of clauses 175 to 182, wherein the ligand conjugate is of the formula BLDx, wherein B is a cell surface receptor targeting ligand, D is an independently selected drug, x is an integer selected from 1, 2, 3, 4 and 5; and L is a releasable polyvalent linker comprising a thiol reactive linkage; or a pharmaceutically acceptable salt thereof.        186. The method or use of clause 185, wherein B is folate.        187. The method or use of clause 185, wherein B is D-folate.        188. The method or use of clause 185, wherein B is L-folate.        189. The method or use of clause 185, wherein B is a PSMA binding ligand.        190. The method or use of clause 185, wherein B is a radical of the formula        
                191. The method or use of clause 185, wherein B is a radical of the formula        
                192. The method or use of clause 185, wherein B is a radical of the formula        
                193. The method or use of any one of clauses 185 to 192, wherein the thiol reactive linkage is a disulfide linkage.        194. The method or use of any one of clauses 185 to 193, wherein L comprises a cysteine disulfide diradical.        195. The method or use of any one of clauses 185 to 194, wherein L further comprises one or more divalent hydrophilic radicals.        196. The method or use of any one of clauses 185 to 195, wherein D is a cytotoxic agent.        197. The method or use of any one of clauses 185 to 196, wherein D is a cancer treating agent.        198. The method or use of any one of clauses 185 to 196, wherein D is an anti-inflammatory agent.        199. The method or use of any one of clauses 185 to 198, wherein D is a vinca alkaloid.        200. The method or use of any one of clauses 185 to 198, wherein D is desacetylvinblastine monohydrazide.        201. The method or use of any one of clauses 185 to 198, wherein D is a tubulysin.        202. The method or use of any one of clauses 185 to 198, wherein D is tubulysin A.        203. The method or use of any one of clauses 185 to 198, wherein D is tubulysin B.        204. The method or use of any one of clauses 185 to 198, wherein D is tubulysin A hydrazide.        205. The method or use of any one of clauses 185 to 198, wherein D is tubulysin B hydrazide.        206. The method or use of any one of clauses 185 to 198, wherein D is an antifolate.        207. The method or use of any one of clauses 185 to 198, wherein D is an aminopterin.        208. The method or use of any one of clauses 185 to 198, wherein D is a rapamycin.        209. The method or use of any one of clauses 185 to 198, wherein D is a mitomycin.        210. The method or use of any one of clauses 185 to 198, wherein D is a taxane.        211. The method or use of any one of clauses 185 to 198, wherein D is a doxorubicin.        212. The method or use of any one of clauses 175 to 183 or 185 to 211, wherein the ligand conjugate is a folate conjugate.        213. The method or use of clause 212, wherein the folate conjugate is        
                214. The method or use of clause 212, wherein the folate conjugate is        
                215. The method or use of any one of clauses 175 to 214, wherein the system xc− inhibitor is selected from the group consisting of sulfasalazine, glutamate; L-quisqualate; (S)-4-carboxyphenylglycine (4-S—CPG); L-α-aminoadipic acid; and L-homocysteic acid.        216. The method or use of any one of clauses 175 to 215, wherein the system xc− inhibitor is sulfasalazine.        217. The method or use of any one of clauses 175 to 215, wherein the system xc− inhibitor is glutamate.        218. The method or use of any one of clauses 175 to 215, wherein the system xc− inhibitor is L-quisqualate.        219. The method or use of any one of clauses 175 to 215, wherein the system xc− inhibitor is 4-S—CPG.        220. The method or use of any one of clauses 175 to 215, wherein the system xc− inhibitor is L-α-aminoadipic acid.        221. The method or use of any one of clauses 175 to 215, wherein the system xc− inhibitor is L-homocysteic acid.        222. The method or use of any one of clauses 175 to 221, wherein the ligand conjugate and the system xc− inhibitor are in parenteral dosage forms.        223. The method or use of clause 222, wherein the dosage forms are independently selected from the group consisting of intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, and intrathecal.        224. The method or use of any one of clauses 175 to 223, wherein the ligand conjugate is in a composition and the system xc− inhibitor is in a composition and wherein the compositions further comprise pharmaceutically acceptable carriers.        225. The method or use of clause 224, wherein the pharmaceutically acceptable carriers are liquid carriers.        226. The method or use of clause 225, wherein the liquid carriers are independently selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof.        227. The method or use of any one of clauses 175 to 226, wherein the ligand conjugate and the system xc− inhibitor are administered in therapeutically effective amounts.        228. The method or use of clause 227, wherein the effective amounts range from about 1 μg/m2 to about 500 mg/m2 of body surface area.        229. The method or use of clause 227, wherein the effective amounts range from about 1 μg/m2 to about 300 mg/m2 of body surface area.        230. The method or use of clause 227, wherein the effective amounts range from about 10 μg/kg to about 100 μg/kg of patient body weight.        231. The method or use of any one of clauses 175 to 230, wherein the ligand conjugate and the system xc− inhibitor are in sterile containers or packages.        232. The method or use of any one of clauses 175 to 231, wherein the ligand conjugate and the system xc− inhibitor have a purity of at least 90% based on weight percentage.        233. The method or use of any one of clauses 178 to 232, wherein the ligand conjugate is in the form of a reconstitutable lyophilizate.        234. The method or use of any one of clauses 175 to 233, wherein the ligand conjugate and the system xc− inhibitor are in sterile, pyrogen-free aqueous solutions.        235. A kit comprising a ligand conjugate and one or more system xc− inhibitors, wherein the ligand conjugate comprises a disulfide linkage.        236. The kit of clause 235, wherein the ligand is folate.        237. The kit of clause 235, wherein the ligand is an antibody.        238. The kit of clause 235, wherein the ligand conjugate is of the formula BLDx, wherein B is a cell surface receptor targeting ligand, D is an independently selected drug, x is an integer selected from 1, 2, 3, 4 and 5; and L is a releasable polyvalent linker comprising a thiol reactive linkage; or a pharmaceutically acceptable salt thereof.        239. The kit of clause 238, wherein B is folate.        240. The kit of clause 238, wherein B is D-folate.        241. The kit of clause 238, wherein B is L-folate.        242. The kit of clause 238, wherein B is a PSMA binding ligand.        243. The kit of clause 238, wherein B is a radical of the formula        
                244. The kit of clause 238, wherein B is a radical of the formula        
                245. The kit of clause 238, wherein B is a radical of the formula        
                246. The kit of any one of clauses 238 to 245, wherein the thiol reactive linkage is a disulfide linkage.        247. The kit of any one of clauses 238 to 246, wherein L comprises a cysteine disulfide diradical.        248. The kit of any one of clauses 238 to 247, wherein L further comprises one or more divalent hydrophilic radicals.        249. The kit of any one of clauses 238 to 248, wherein D is a cytotoxic agent.        250. The kit of any one of clauses 238 to 249, wherein D is a cancer treating agent.        251. The kit of any one of clauses 238 to 249, wherein D is an anti-inflammatory agent.        252. The kit of any one of clauses 238 to 251, wherein D is a vinca alkaloid.        253. The kit of any one of clauses 238 to 251, wherein D is desacetylvinblastine monohydrazide.        254. The kit of any one of clauses 238 to 251, wherein D is a tubulysin.        255. The kit of any one of clauses 238 to 251, wherein D is tubulysin A.        256. The kit of any one of clauses 238 to 251, wherein D is tubulysin B.        257. The kit of any one of clauses 238 to 251, wherein D is tubulysin A hydrazide.        258. The kit of any one of clauses 238 to 251, wherein D is tubulysin B hydrazide.        259. The kit of any one of clauses 238 to 251, wherein D is an antifolate.        260. The kit of any one of clauses 238 to 251, wherein D is an aminopterin.        261. The kit of any one of clauses 238 to 251, wherein D is a rapamycin.        262. The kit of any one of clauses 238 to 251, wherein D is a mitomycin.        263. The kit of any one of clauses 238 to 251, wherein D is a taxane.        264. The kit of any one of clauses 238 to 251, wherein D is a doxorubicin.        265. The kit of any one of clauses 235 to 236 or 238 to 264, wherein the ligand conjugate is a folate conjugate.        266. The kit of clause 265, wherein the folate conjugate is        
                267. The kit of clause 265, wherein the folate conjugate is        
                268. The kit of any one of clauses 235 to 267, wherein the system xc− inhibitor is selected from the group consisting of sulfasalazine, glutamate; L-quisqualate; (S)-4-carboxyphenylglycine (4-S—CPG); L-α-aminoadipic acid; and L-homocysteic acid.        269. The kit of any one of clauses 235 to 268, wherein the system xc− inhibitor is sulfasalazine.        270. The kit of any one of clauses 235 to 268, wherein the system xc− inhibitor is glutamate.        271. The kit of any one of clauses 235 to 268, wherein the system xc− inhibitor is L-quisqualate.        272. The kit of any one of clauses 235 to 268, wherein the system xc− inhibitor is 4-S—CPG.        273. The kit of any one of clauses 235 to 268, wherein the system xc− inhibitor is L-α-aminoadipic acid.        274. The kit of any one of clauses 235 to 268, wherein the system xc− inhibitor is L-homocysteic acid.        275. The kit of any one of clauses 235 to 274, wherein the ligand conjugate and the system xc− inhibitor are in parenteral dosage forms.        276. The kit of clause 275, wherein the dosage forms are independently selected from the group consisting of intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, and intrathecal.        277. The kit of any one of clauses 235 to 276, wherein the ligand conjugate is in a composition and the system xc− inhibitor is in a composition and wherein the compositions further comprise pharmaceutically acceptable carriers.        278. The kit of clause 277, wherein the pharmaceutically acceptable carriers are liquid carriers.        279. The kit of clause 278, wherein the liquid carriers are independently selected from the group consisting of saline, glucose, alcohols, glycols, esters, amides, and a combination thereof.        280. The kit of any one of clauses 235 to 279, wherein the ligand conjugate and the system xc− inhibitor are in therapeutically effective amounts.        281. The kit of any one of clauses 235 to 280, wherein the ligand conjugate and the system xc− inhibitor are in sterile containers or packages.        282. The kit of any one of clauses 235 to 281, wherein the ligand conjugate and the system xc− inhibitor have a purity of at least 90% based on weight percentage.        283. The kit of any one of clauses 235 to 281, wherein the ligand conjugate and the system xc− inhibitor have a purity of at least 95% based on weight percentage.        284. The kit of any one of clauses 235 to 283, wherein the ligand conjugate is in the form of a lyophilizate.        285. The kit of any one of clauses 235 to 284, wherein the ligand conjugate is in the form of a reconstitutable lyophilizate.        286. The kit of any one of clauses 235 to 285, wherein the ligand conjugate and the system xc− inhibitor are in sterile, pyrogen-free aqueous solutions.        287. An in vitro assay for identifying a ligand conjugate suitable for co-administration to a patient with a system xc− inhibitor, the assay comprising:        a) adding the ligand conjugate to the culture medium of a first sample of cultured cells, wherein the ligand conjugate comprises a disulfide linkage;        b) adding the system xc− inhibitor to the culture medium of the first sample of cultured cells to provide a test sample;        c) adding the ligand conjugate to the culture medium of a second sample of cultured cells to provide a control sample;        d) measuring the non-ligand-specific activity of the ligand conjugate or the nonspecific uptake of the drug in the test sample;        e) measuring the non-ligand-specific activity of the ligand conjugate or the nonspecific uptake of the drug in the control sample; and        f) determining that the ligand conjugate is suitable for co-administration to the patient with the system xc− inhibitor if the non-ligand-specific activity of the ligand conjugate and/or the nonspecific uptake of the drug are decreased in the test sample relative to the control sample.        288. The in vitro assay of clause 287 further comprising step g) administering the ligand conjugate and the system xc− inhibitor to the patient.        289. The in vitro assay of clause 287 or clause 288 wherein the ligand is folate.        290. The in vitro assay of clause 287 or clause 288 wherein the ligand is an antibody.        291. The in vitro assay of clause 287 or clause 288, wherein the ligand conjugate is of the formula BLDx, wherein B is a cell surface receptor targeting ligand, D is an independently selected drug, x is an integer selected from 1, 2, 3, 4 and 5; and L is a releasable polyvalent linker comprising a thiol reactive linkage; or a pharmaceutically acceptable salt thereof.        292. The in vitro assay of clause 291, wherein B is folate.        293. The in vitro assay of clause 291, wherein B is D-folate.        294. The in vitro assay of clause 291, wherein B is L-folate.        295. The in vitro assay of clause 291, wherein B is a PSMA binding ligand.        296. The in vitro assay of clause 291, wherein B is a radical of the formula        
                297. The in vitro assay of clause 291, wherein B is a radical of the formula        
                298. The in vitro assay of clause 291, wherein B is a radical of the formula        
                299. The in vitro assay of any one of clauses 291 to 298, wherein the thiol reactive linkage is a disulfide linkage.        300. The in vitro assay of any one of clauses 291 to 299, wherein L comprises a cysteine disulfide diradical.        301. The in vitro assay of any one of clauses 291 to 300, wherein L further comprises one or more divalent hydrophilic radicals.        302. The in vitro assay of any one of clauses 291 to 301, wherein D is a cytotoxic agent.        303. The in vitro assay of any one of clauses 291 to 302, wherein D is a cancer treating agent.        304. The in vitro assay of any one of clauses 291 to 302, wherein D is an anti-inflammatory agent.        305. The in vitro assay of any one of clauses 291 to 304, wherein D is a vinca alkaloid.        306. The in vitro assay of any one of clauses 291 to 304, wherein D is desacetylvinblastine monohydrazide.        307. The in vitro assay of any one of clauses 291 to 304, wherein D is a tubulysin.        308. The in vitro assay of any one of clauses 291 to 304, wherein D is tubulysin A.        309. The in vitro assay of any one of clauses 291 to 304, wherein D is tubulysin B.        310. The in vitro assay of any one of clauses 291 to 304, wherein D is tubulysin A hydrazide.        311. The in vitro assay of any one of clauses 291 to 304, wherein D is tubulysin B hydrazide.        312. The in vitro assay of any one of clauses 291 to 304, wherein D is an antifolate.        313. The in vitro assay of any one of clauses 291 to 304, wherein D is an aminopterin.        314. The in vitro assay of any one of clauses 291 to 304, wherein D is a rapamycin.        315. The in vitro assay of any one of clauses 291 to 304, wherein D is a mitomycin.        316. The in vitro assay of any one of clauses 291 to 304, wherein D is a taxane.        317. The in vitro assay of any one of clauses 291 to 304, wherein D is a doxorubicin.        318. The in vitro assay of any one of clauses 287 to 290, wherein the ligand conjugate is a folate conjugate.        319. The in vitro assay of clause 318, wherein the folate conjugate is        
                320. The in vitro assay of clause 318, wherein the folate conjugate is        
                321. The in vitro assay of any one of clauses 287 to 320, wherein the system xc− inhibitor is selected from the group consisting of sulfasalazine, glutamate; L-quisqualate; (S)-4-carboxyphenylglycine (4-S—CPG); L-α-aminoadipic acid; and L-homocysteic acid.        322. The in vitro assay of any one of clauses 287 to 321, wherein the system xc− inhibitor is sulfasalazine.        323. The in vitro assay of any one of clauses 287 to 321, wherein the system xc− inhibitor is glutamate.        324. The in vitro assay of any one of clauses 287 to 321, wherein the system xc− inhibitor is L-quisqualate.        325. The in vitro assay of any one of clauses 287 to 321, wherein the system xc− inhibitor is 4-S—CPG.        326. The in vitro assay of any one of clauses 287 to 321, wherein the system xc− inhibitor is L-α-aminoadipic acid.        327. The in vitro assay of any one of clauses 287 to 321, wherein the system xc− inhibitor is L-homocysteic acid.        328. The in vitro assay of any one of clauses 287 to 327, wherein the cultured cells are KB cells.        329. The in vitro assay of any one of clauses 287 to 327, wherein the cultured cells are A549 cells.        330. The in vitro assay of any one of clauses 287 to 329, wherein the non-ligand-specific activity of the ligand conjugate is decreased by the system xc− inhibitor.        331. The in vitro assay of clause 330, wherein the non-ligand-specific activity is cytotoxicity.        332. The in vitro assay of any one of clauses 287 to 330, wherein the nonspecific uptake of the drug is decreased by the system xc− inhibitor.        333. The in vitro assay of clause 332, wherein the nonspecific uptake of the drug is measured using competition assays in the presence and absence of an excess of non-radiolabeled ligand.        